AAV‐mediated delivery of an anti‐BACE1 VHH alleviates pathology in an Alzheimer's disease model

Abstract Single domain antibodies (VHHs) are potentially disruptive therapeutics, with important biological value for treatment of several diseases, including neurological disorders. However, VHHs have not been widely used in the central nervous system (CNS), largely because of their restricted blood–brain barrier (BBB) penetration. Here, we propose a gene transfer strategy based on BBB‐crossing adeno‐associated virus (AAV)‐based vectors to deliver VHH directly into the CNS. As a proof‐of‐concept, we explored the potential of AAV‐delivered VHH to inhibit BACE1, a well‐characterized target in Alzheimer’s disease. First, we generated a panel of VHHs targeting BACE1, one of which, VHH‐B9, shows high selectivity for BACE1 and efficacy in lowering BACE1 activity in vitro. We further demonstrate that a single systemic dose of AAV‐VHH‐B9 produces positive long‐term (12 months plus) effects on amyloid load, neuroinflammation, synaptic function, and cognitive performance, in the AppNL‐G‐F Alzheimer’s mouse model. These results constitute a novel therapeutic approach for neurodegenerative diseases, which is applicable to a range of CNS disease targets.

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*Additional important information regarding figures and illustrations can be found at http://bit.ly/EMBOPressFigurePreparationGuideline ***** Reviewer's comments ***** Referee #1 (Remarks for Author): In this manuscript titled "AAV mediated delivery of a novel anti-BACE1 VHH reduces Abeta in an Alzheimer's disease mouse model", Melvin Y. Rincon et al develop a novel VHH (VHH-B9) to inhibit BACE1 activity in vitro and propose a gene transfer strategy to deliver this VHH directly into CNS by means of using AAV as a vector. In vivo injections of AAV encoded VHH-B9 demonstrated reductions in amyloid beta levels in mice model of cerebral amyloidosis. Similar approach using AAV delivered nanobody in different murine model has been tested (Verhelle A., et al., Human Molecular Genetics, 2017). The main weakness of this study is the lack of evidences for therapeutic benefits in the treated animals. More comprehensive analyses will strengthen the story.
Major points: 1. Lack of data to show the improvements in pathology and cognitive function in treated animals. 2. The authors did not check for long term persistence of VHH-B9 expression in mice brain. Mice were sacked 3 weeks post injection. For therapeutic purposes, considering the route of administration is intracranial, the timelines are much longer, and repeated administration is inconvenient and at times not feasible, monitoring VHH-B9 levels at later time points is important to conclude that there is lack of clearance. 3. Did the authors observe any physical manifestations of the reduction in A 1-40 and A 1-42 in mice brain like reduction in neuroinflammation or hemorrhages which are typically seen in APP Dutch mice?
Minor points: 1. Why did the authors select AAV1 as the preferred serotype for in vivo administration over some other serotypes like AAV9 or PHP.B that have been demonstrated in the past to have higher transduction efficiency for brain than AAV1? 2. The author should examine for any immune responses against either the AAV or VHH-B9. They can conduct simple ELISAs to look for antibodies against the transgene. 3. The most important data presented in this manuscript is Figure 3E and 3F. However, the authors claimed for a significant reduction of A 1-40 and A 1-42 in animals without any statistical analysis shown in the data figure. This is totally unacceptable.

Referee #2 (Comments on Novelty/Model System for Author):
This is an interesting study with important therapeutic implications for neurodegenerative disease including Alzheimer's disease. The authors do a thorough job characterizing VHH-B9 and testing its efficacy in vitro and in vivo, and the manuscript is well written.
Referee #2 (Remarks for Author): In this study, the authors engineer single domain antibodies from camel to recognize the beta-secretase BACE1 as a proof of concept for the development of CNS therapeutics. One single domain antibody, VHH-B9, binds to an exosite of BACE1 with nanomolar affinity, does not bind the close homolog BACE2, and inhibits processing of APP and Abeta generation in cell-free assays and in primary neuron cultures. Interestingly, VHH-B9 inhibits BACE1 cleavage of APP but not another BACE1 substrate Sez6. The authors then generate an AAV-VHH-B9 vector and show that following hippocampal injection APP-dutch mice exhibit co-localization of VHH-B9 and BACE1 in intracellular compartments and show significantly reduced Abeta 40 and Abeta42 production in the brain. The authors conclude that AAV delivery of engineered single domain antibodies constitute a novel therapeutic approach for targeting a wide range of CNS targets for neurodegenerative diseases. This is an interesting study with important therapeutic implications for neurodegenerative disease including Alzheimer's disease.
1. The authors explanation as to why VHH-B9 inhibits BACE1 cleavage of APP but not Sez6 should be elaborated in the discussion. The statement that the substrate selectivity "is most simply explained by the relative amounts of APP and SEZ6 available for cleavage in a mass-action model (Wilhelm et al, 2014)" is rather vague. In addition, it is not immediately clear why compound J would not behave in a similar fashion in such a mass-action model. Other explanations may exist and should be explored, as an APP-selective BACE1 inhibitor would be enormously valuable for avoiding substrate-specific side effects. 2. It is unclear why APP-dutch mice were used instead of a more commonly used APP transgenic model. The authors should explain their rationale for this mouse. 3. Did the authors explore amyloid pathology by immunostainng? If so, please include some Abeta stained sections.
Referee #3 (Comments on Novelty/Model System for Author): The model systems used so far in this manuscript are adequate. However, I think the authors would need to go a bit further to identify potential functional impact of their promising approach. This could be achieved in a disease mouse model with clear behavioral and neuropathological defects.

Referee #3 (Remarks for Author):
This is an interesting piece of work aimed at generating single domain antibodies (VHH) for therapeutic purposes. The team identified an efficient VHH-B9 following immunization of dromedary and a Ilama. The VHH generated is targeting the beta-site amyloid precursor protein cleavage enzym 1 (BACE1). The VHH-B9 cDNA was packaged in a AAV vector system for potential gene transfer in vitro and in vivo. They nicely demonstrated that VHH-B9 mediated selective lowering of BACE1 activity in vitro, including neuronal cells. They also report that VHH-B9 led to reduced levels of amyloid beta (Ab) following AAV-mediated delivery of VHH-B9 into CNS of a mouse model of cerebral amyloidosis. This is an exciting approach with some evidence of efficacy in vitro and in vivo. However, it would be very compelling if the strategy is leading to functional recovery in a mouse model of disease. This could be achieved by measuring impact on behavioral and neuropathological defects. Including such data could take this manuscript to a high and convincing level.
Minor comments: 1) what dose of AAV was delivered in mice? 2) Page 6: line 1: please refer to the correct figure 2 instead to Figure 1. 3) Last paragraph of result section claim no toxicity was observed following delivery in brain of mice. How measurement of toxicity was achieved. Please elaborate further 4) Figure 3F: Y axis description is missing

EMM-2018-09824: Marino et al.: Point-by-point response to the reviewers' comments:
We thank all of the reviewers for highlighting the importance of our findings and the clinical potential.
On the advice of the reviewers, we have added substantial experimental data, which can be summarized as follows: 1) Use of an alternative blood brain barrier crossing AAV capsid, PHP.B, allowing intravenous delivery in a next generation Alzheimer's model, App NL-G-F .
2) A long-term, integrated study combining behavioral analysis, to assess possible cognitive improvements in App NL-G-F mice following AAV-VHH-B9 administration, with subsequent post-mortem analysis of Ab levels, associated neuroinflammation and neuronal circuit performance.
We greatly appreciated the reviewers' critiques. We believe that by addressing these, we have substantially improved the manuscript. As a result, we have completely redrafted the text using the 'Research Article' format, which we believe is more suited to allow clear presentation and discussion of the additional data generated during the revision period. Below, we provide answers to all the individual concerns raised during the initial review.

Referee #1 (Remarks for Author):
In this manuscript titled "AAV mediated delivery of a novel anti-BACE1 VHH reduces Abeta in an We thank the reviewer for his/her insightful comments and believe they have helped us significantly improve the manuscript.
Major points:

Lack of data to show the improvements in pathology and cognitive function in treated animals.
We agree with the reviewer that lack of data showing long-term cognitive improvement was a weakness in the original submission. Therefore, we have undertaken a comprehensive set of experiments which show that long-term VHH-B9 expression, following AAV-based delivery, preserves aspects of neuronal circuit function (LTP) ( Figure 5) and preserves cognitive function in a 'Sociability and Preference for 6th Nov 2021 1st Authors' Response to Reviewers Social Novelty' paradigm ( Figure 2B). These improvements are associated with significant reductions in Ab1-40 and Ab1-42 levels (Figure 3), reduced astro-and microgliosis ( Figure 4) and reduced synaptic loss ( Figure EV5B). 3. Did the authors observe any physical manifestations of the reduction in Ab1-40 and Ab1-42 in mice brain like reduction in neuroinflammation or hemorrhages which are typically seen in APP Dutch mice?

The authors did not check for long term persistence of VHH
We changed our mouse model to the App NL-G-F line, in response to a comment from Reviewer 2. We chose the App NL-G-F line as it is a knock-in model (Saito et al, Nat Neurosci, 2014) and more likely recapitulates endogenous patterns of APP expression and Ab production. In this model, we observed that reduction of Ab1-40 and Ab1-42, following VHH-B9 treatment, was associated with reduced neuroinflammation, reduced synaptic loss and improved plasticity of neuronal circuits.

Minor points:
1. Why did the authors select AAV1 as the preferred serotype for in vivo administration over some other serotypes like AAV9 or PHP.B that have been demonstrated in the past to have higher transduction efficiency for brain than AAV1?
The choice of AAV1 as the preferred serotype for direct intraparenchymal injections was largely based on our previous successful use of AAV1 in such protocols. However, in this revised manuscript, we have switched to PHP.B, as this serotype has been shown to give high levels of CNS transduction following intravenous administration, which we believe is a more therapeutically relevant delivery method.
2. The author should examine for any immune responses against either the AAV or VHH-B9. They can conduct simple ELISAs to look for antibodies against the transgene.
We thank the reviewer for this suggestion.
We performed an ELISA to investigate the possible immune response against VHH-B9, and this new data is given in Appendix Figure 2. Under the experimental conditions used, we could not detect a significant immune (humoral) response against VHH-B9, consistent with the known low immunogenicity of VHH. We believe this explains the fact that we see long-term VHH expression in our experiments, in the absence of overt toxicity ( Figure 2C), as well as reduced pathology in App NL-G-F mice.
We did not perform experiments designed to detect immune response to AAV, as anti-capsid antibodies are known to be rapidly generated following systemic administration (Verdera et al, Mol Ther, 2020). Figure 3E and 3F. However, the authors claimed for a significant reduction of Ab1-40 and Ab1-42 in animals without any statistical analysis shown in the data figure. This is totally unacceptable.

The most important data presented in this manuscript is
We apologize for this oversight. As it stands, we have now replaced the offending figure with new data on Ab1-40 and Ab1-42 reduction following intravenous delivery of AAV.PHP.B-VHH-B9, with appropriate statistical analysis (Figure 3). We have also double-checked other figures in the revised manuscript to ensure that statistical analysis is included where appropriate. We hope this satisfies the reviewer.

Referee #2 (Comments on Novelty/Model System for Author):
This is an interesting study with important therapeutic implications for neurodegenerative disease including Alzheimer's disease. The authors do a thorough job characterizing VHH-B9 and testing its efficacy in vitro and in vivo, and the manuscript is well written.
We thank the reviewer for their positive assessment of our work and recognition of its importance.

Referee #2 (Remarks for Author):
In this study, the authors engineer single domain antibodies from camel to recognize the beta-secretase We thank the reviewer for raising this issue.
We agree that an APP-selective BACE1 inhibitor would be of enormous value for avoiding substratespecific side effects. We respectfully point out, however, that the main focus of the paper is on providing proof-of-concept for AAV-mediated nanobody delivery for long-term treatment of CNS disease. In this respect, we choose BACE1 as a target for nanobody production, as it has proven to be an ideal target for exploring the potential of antibody-based therapeutics in the CNS (Atwal et al., Sci Transl Med, 2011). BACE1 is constitutively active, accounting for the majority of Aβ production in vivo. Therefore, measuring Aβ concentrations after anti-BACE1 administration provides a direct measure of antibodymediated (or nanobody-mediated) target neutralization.
As such, we decided not to emphasize the issue of substrate specificity in the current paper, particularly as we only test one known BACE1 substrate, Sez6. Hence, we have moved these blots to the 'Appendix' and restrict mention of the therapeutic potential of such specific inhibitors to the 'Discussion'.
Our intention is to follow up this (important) observation in a separate project, in which we can explore potential APP specificity by assessing cleavage of a wide range of alternate BACE1 substrates (Barāo

It is unclear why APP-dutch mice were used instead of a more commonly used APP transgenic model.
The authors should explain their rationale for this mouse.
Our reason for using the APP-Dutch mouse line was that we had previously used it to show that the anti-BACE1 monoclonal antibody 1A11 inhibits Aβ production in vivo, following direct injection into the hippocampus (Zhou et al, J Biol Chem, 2011). Hence, we regarded it as an ideal control platform to test the activity of our newly generated anti-BACE1 nanobodies. At the time this particular set of experiments was performed, we also had animals of the right age immediately available for injection.
However, we acknowledge the concerns of the reviewer. As both Reviewers 1 and 3 asked to see a functional impact of VHH-B9 delivery in an Alzheimer's model, we needed to run a set of long-term behavior experiments during the review period. Therefore, we took this opportunity to switch to the commonly used, and well characterized, App NL-G-F knock in line (Saito et al, Nat Neurosci, 2014).

Did the authors explore amyloid pathology by immunostaining? If so, please include some Abeta stained sections.
The requested stainings are now included (Figure 3 and Figure EV5A).

Referee #3 (Comments on Novelty/Model System for Author):
The model systems used so far in this manuscript are adequate. However, I think the authors would need to go a bit further to identify potential functional impact of their promising approach. This could be achieved in a disease mouse model with clear behavioral and neuropathological defects.
We thank the reviewer for their constructive critique of our work. In our revised manuscript, we have acted on these comments and show a clear functional impact of our technology in the App NL-G-F mouse line, at both neuropathological and behavioral levels.

This is an interesting piece of work aimed at generating single domain antibodies (VHH) for therapeutic purposes. The team identified an efficient VHH-B9 following immunization of dromedary and a Ilama.
The VHH generated is targeting the beta-site amyloid precursor protein cleavage enzyme 1 (BACE1).

The VHH-B9 cDNA was packaged in a AAV vector system for potential gene transfer in vitro and in vivo. They nicely demonstrated that VHH-B9 mediated selective lowering of BACE1 activity in vitro, including neuronal cells. They also report that VHH-B9 led to reduced levels of amyloid beta (Ab) following AAV-mediated delivery of VHH-B9 into CNS of a mouse model of cerebral amyloidosis.
This is an exciting approach with some evidence of efficacy in vitro and in vivo. However, it would be very compelling if the strategy is leading to functional recovery in a mouse model of disease. This could be achieved by measuring impact on behavioral and neuropathological defects. Including such data could take this manuscript to a high and convincing level.
As indicated above, during revision, we have added the neuropathological and behavioral data requested.

1) what dose of AAV was delivered in mice?
We have now switched to intravenous delivery of AAV, rather than using direct parenchymal injection.
Using tail vein injection, we routinely administer 1x10 12 vector genomes (vg) in a total solution volume of 100 µl per mouse. We have included this information in the main text and figure legend describing the behavioral experiments performed (Figure 2). Follow up experiments measuring Ab levels, associated neuroinflammation and neuronal circuit performance (Figures 3-5), were all performed postmortem on tissue recovered from these mouse cohorts (a detail highlighted in the text). During the revision period, we have added substantial new data to the manuscript to satisfy the reviewers' comments. This has led to a restructuring of the text. We have taken great care to ensure that all figures are correctly referenced in this resubmitted version.

How measurement of toxicity was achieved. Please elaborate further
Here, we were referring to the fact that vector administration and nanobody administration did not lead to abnormal behavior, nor premature death, in the injected mice.
In our revised manuscript, we have performed long-term experiments and shown that this lack of overt toxicity holds 12 months post-injection. We have added this information to the text as Appendix Table   1, containing full details of the mouse cohorts used. To avoid any confusion as to what we mean by 'toxicity' we now refer specifically to 'behavioral abnormalities' and 'mortality rate'. We hope this satisfactorily addresses the issue. Figure 3F: Y axis description is missing

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The measurement of Ab1-42 has been repeated following intravenous administration of AAV.PHP.B-VHH-B9 ( Figure 3D). We have made sure that the graph axes are correctly labeled. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed report from two of the three referees who agreed to re-assess it. Unfortunately, after a series of reminders, we did not obtain a report from Referee #3. In the interest of time, and since the other two referees' recommendations are similar, I prefer to make a decision now rather than further delay the process. As you will see, the referees are now overall supportive, and I am pleased to inform you that we will be able to accept your manuscript pending the following amendments: https://www.embopress.org/doi/pdf/10.1002/emmm.201000094), EMBO Molecular Medicine will publish online a Review Process File to accompany accepted manuscripts.
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Two sets of experiments were performed, an initial proof-of-concept (PoC) to test VHH production and target engagement following systemic (tail vein) AAV injection, and a longitudinal study (BEHAVIOR) to assess the possible beneficial effects of extended VHH expression/activity. Both sets of experiments were performed in the AppNL-G-F mouse line. PoC: Experiments using cultured neurons ( Figure 1D) showed a significant reduction in amyloid beta production following VHH-B9 administration. Based on this data, we designed PoC experiments assuming an effect size of 2 and assuming 80% power and an alpha of 0.05, giving a minimum of six animals in each treatment cohort. BEHAVIOR: Based on previous experiments with the AppNL-G-F line, an effect size of 1.36 was expected (Latif-Hernandez et al. (2019) Subtle behavioral changes and increased prefrontalhippocampal synchronicity in AppNL-G-F mice before dominant plaque deposition. Behav Brain Res, 17: 431-441). Assuming 90% power and an alpha of 0.05, a minimal sample size of n=13 per group was calculated. However, to offset possible loss of animals due to aging-related issues (including potential long-term effects of vector injections and/or onset of AD-type pathology), we added n=3-5 to the starting groups, to account for potential losses. Similar sample size was adopted for the C57Bl/6J controls ( Figure Legends; Appendix Table S1) graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
PoC: Sample size estimates were made based on reductions in amyloid beta production seen after testing VHH-B9 efficacy in tissue culture experiments ( Figure 1D). BEHAVIOR: The sample size for our long-term animal study was estimated based on previous observations with the AppNL-G-F line, taking into account the necessity to perform behavioural studies in aged animals, with overt AD pathology and viral vector treatment both potentially contributing to premature death (relative to BL6 controls).
PoC: No animals were excluded from the analysis. BEHAVIOR: Animals were excluded in the case of technical problems (i.e. incorrect tracking). In addition, non-performers were removed from analysis of data obtained using the Morris Water Maze, based on pre-established criteria (swim velocity < 5cm/s for longer than 35% of total time).
PoC and BEHAVIOR: Particular care was put into avoiding any potential subjective bias during allocation of the animals into treatment groups; Assignment to AAV treatment groups was performed randomly. PoC: Both male and female animals were used (Appendix Table S1). No blinding was performed in respect of the vector treatments given to each experimental cohort. BEHAVIOR: To avoid any potential gender-dependent effects, all the animals included in the longterm study were males. During vector administration, the scientist performing injections was blinded to the AAV-type being administered. The C57Bl/6J non-injected group was simply established using the n=18 animals that were left over following AAV vector administration, generating a comparable control group.

Manuscript Number: EMM-2018-09824
PoC and BEHAVIOR: Allocation of the animals into each treatment group was performed randomly. BEHAVIOR: the order the animals were used in testing was randomised and the tester was blinded to treatment group.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.